Detection of endogenous Snf1 and its activation state: application to Saccharomyces and Candida species.
The stress-response Snf1 protein kinase of Saccharomyces cerevisiae serves as a powerful model for studies of the eukaryotic Snf1/AMP-activated protein kinase (AMPK) family. Central to studies of Snf1 are methods that determine its activation state under various physiological and genetic conditions. Here, we have developed a convenient and sensitive method for immunoblot analysis of endogenous yeast Snf1 and its activation-loop threonine (Thr210) phosphorylation. The method employs readily obtainable reagents and yields results that faithfully reflect the environmental and genetic conditions tested. Using our method, we have obtained evidence that Snf1 remains stress-regulated in reg1 Delta cells, revealing the existence of a Snf1 signalling mechanism(s) that is independent of Reg1-PP1 phosphatase. In addition to strains of common laboratory S. cerevisiae backgrounds, we have applied the method to two pathogenic Candida species, C. glabrata and C. albicans. We have detected proteins whose gel mobilities, immune properties and regulation patterns are consistent with those expected for the corresponding Snf1 homologues. Because Snf1 activation is a sensitive marker of several types of stress, including artifactual stresses associated with common cell harvesting and protein extraction procedures, the convenient and efficient protein extraction method described here should be advantageous for SDS-PAGE and immunoblot analyses of stress-regulated and other proteins from various yeast species.
Orlova
Marianna
M
Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, USA.
Barrett
LaKisha
L
Kuchin
Sergei
S
D002175:Candida / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation; D015966:Gene Expression Regulation, Fungal; D015151:Immunoblotting / Q000379:methods*; D009154:Mutation; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D012440:Saccharomyces / Q000201:enzymology* / Q000235:genetics; D012680:Sensitivity and Specificity; D015398:Signal Transduction
C071570:SNF1-related protein kinases; D017346:Protein Serine-Threonine Kinases